Surprisingly little is known about how proteins fold in vivo, yet it is this process, and not the test-tube[unreadable] idealized folding reaction so intensively studied over the past several decades, that is crucial to the[unreadable] fitness of an organism. The fidelity of folding and the stability of proteins in the cell are critical to their[unreadable] functions, their degradation, and their vulnerability to aggregation. Many diseases are now known to[unreadable] arise from defects in protein folding, either because of the loss or alteration of essential protein[unreadable] functions, or because of the build-up of toxic species such as aggregates. We believe that novel[unreadable] methods and creative collaborations will allow us to overcome the daunting technical obstacles that[unreadable] have impeded progress on protein folding in the cell. Focusing first on a small model protein for which[unreadable] we have detailed descriptions of folding in vitro will enable methods optimization. Folding in cellular[unreadable] conditions will be followed in systems of increasing complexity: bacterial protein expression, cell-free[unreadable] biosynthesis, and semi-permeabilized or intact eukaryotic cell expression. The new strategies will[unreadable] reveal how larger proteins of biomedical interest adopt their structures in their cellular context and how[unreadable] this process may go awry. Methodologically, we anticipate placing major reliance on spectroscopic[unreadable] methods, including fluorescence and nuclear magnetic resonance, and using novel labeling strategies[unreadable] to observe the protein under study in the complex cellular milieu. Complementary in-cell imaging[unreadable] methods will be used to insure that observed signals report on relevant phenomena and to reveal novel[unreadable] functionally significant spatial localization patterns. We anticipate that this research will lead to new[unreadable] paradigms for how amino acid sequences encode folding information and that the resulting enhanced[unreadable] understanding of folding in vivo will lead to new strategies for therapeutic intervention in misfolding and[unreadable] aggregation diseases.